In-gel staining methods of G4 DNA and RNA structures.

G-quadruplexes (G4) are functionally important nucleic acid structures, involved in many cellular pathways. They are often dynamically regulated in cells, which makes detecting them in vivo challenging and dependent on sophisticated technical equipment. Therefore, in vitro studies are commonly performed as a first step to confirm a candidate sequence folds into a G4. Several methods have been developed, each with its individual pros and cons. A highly accessible and quick approach, without the need for specialized equipment, is the detection of G4s in native gels using light-up probes. These molecules become fluorescent after specifically binding to G4s. Several different classes have been discovered, emitting light in various colors, and some possess specificity for certain G4 topologies, which makes them highly versatile tools for G4 visualization. Here, we will explore the general procedure using the light-up probe NMM on RNA G4s and discuss advantages and limitations of this method.

TGF-ß specifies TFH versus TH17 cell fates in murine CD4+ T cells through c-Maf.

T follicular helper (TFH) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse TFH cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor-ß (TGF-ß) induces robust expression of TFH hallmark molecules CXCR5 and Bcl6 in activated mouse CD4+ T cells in vitro. TGF-ß-induced mouse CXCR5+ TFH cells are phenotypically, transcriptionally, and functionally similar to in vivo-generated TFH cells and provide critical help to B cells. The study further reveals that TGF-ß-induced CXCR5 expression is independent of Bcl6 but requires the transcription factor c-Maf. Classical TGF-ß-containing T helper 17 (TH17)-inducing conditions also yield separate CXCR5+ and IL-17A-producing cells, highlighting shared and distinct cell fate trajectories of TFH and TH17 cells. We demonstrate that excess IL-2 in high-density T cell cultures interferes with the TGF-ß-induced TFH cell program, that TFH and TH17 cells share a common developmental stage, and that c-Maf acts as a switch factor for TFH versus TH17 cell fates in TGF-ß-rich environments in vitro and in vivo.

Chronic endoplasmic reticulum stress in myotonic dystrophy type 2 promotes autoimmunity via mitochondrial DNA release.

Myotonic dystrophy type 2 (DM2) is a tetranucleotide CCTG repeat expansion disease associated with an increased prevalence of autoimmunity. Here, we identified an elevated type I interferon (IFN) signature in peripheral blood mononuclear cells and primary fibroblasts of DM2 patients as a trigger of chronic immune stimulation. Although RNA-repeat accumulation was prevalent in the cytosol of DM2-patient fibroblasts, type-I IFN release did not depend on innate RNA immune sensors but rather the DNA sensor cGAS and the prevalence of mitochondrial DNA (mtDNA) in the cytoplasm. Sublethal mtDNA release was promoted by a chronic activation of the ATF6 branch of the unfolded protein response (UPR) in reaction to RNA-repeat accumulation and non-AUG translated tetrapeptide expansion proteins. ATF6-dependent mtDNA release and resulting cGAS/STING activation could also be recapitulated in human THP-1 monocytes exposed to chronic endoplasmic reticulum (ER) stress. Altogether, our study demonstrates a novel mechanism by which large repeat expansions cause chronic endoplasmic reticulum stress and associated mtDNA leakage. This mtDNA is, in turn, sensed by the cGAS/STING pathway and induces a type-I IFN response predisposing to autoimmunity. Elucidating this pathway reveals new potential therapeutic targets for autoimmune disorders associated with repeat expansion diseases.

DNA damage signaling in Drosophila macrophages modulates systemic cytokine levels in response to oxidative stress.

Environmental factors, infection, or injury can cause oxidative stress in diverse tissues and loss of tissue homeostasis. Effective stress response cascades, conserved from invertebrates to mammals, ensure reestablishment of homeostasis and tissue repair. Hemocytes, the Drosophila blood-like cells, rapidly respond to oxidative stress by immune activation. However, the precise signals how they sense oxidative stress and integrate these signals to modulate and balance the response to oxidative stress in the adult fly are ill-defined. Furthermore, hemocyte diversification was not explored yet on oxidative stress. Here, we employed high-throughput single nuclei RNA-sequencing to explore hemocytes and other cell types, such as fat body, during oxidative stress in the adult fly. We identified distinct cellular responder states in plasmatocytes, the Drosophila macrophages, associated with immune response and metabolic activation upon oxidative stress. We further define oxidative stress-induced DNA damage signaling as a key sensor and a rate-limiting step in immune-activated plasmatocytes controlling JNK-mediated release of the pro-inflammatory cytokine unpaired-3. We subsequently tested the role of this specific immune activated cell stage during oxidative stress and found that inhibition of DNA damage signaling in plasmatocytes, as well as JNK or upd3 overactivation, result in a higher susceptibility to oxidative stress. Our findings uncover that a balanced composition and response of hemocyte subclusters is essential for the survival of adult Drosophila on oxidative stress by regulating systemic cytokine levels and cross-talk to other organs, such as the fat body, to control energy mobilization.

TMPRSS2 isoform 1 downregulation by G-quadruplex stabilization induces SARS-CoV-2 replication arrest.

BACKGROUND: SARS-CoV-2 infection depends on the host cell factors angiotensin-converting enzyme 2, ACE2, and the transmembrane serinprotease 2, TMPRSS2. Potential inhibitors of these proteins would be ideal targets against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection. Our data opens the possibility that changes within TMPRSS2 can modulate the outcome during a SARS-CoV-2 infection. RESULTS: We reveal that TMPRSS2 acts not only during viral entry but has also an important role during viral replication. In addition to previous functions for TMPRSS2 during viral entry, we determined by specific downregulation of distinct isoforms that only isoform 1 controls and supports viral replication. G-quadruplex (G4) stabilization by chemical compounds impacts TMPRSS2 gene expression. Here we extend and in-depth characterize these observations and identify that a specific G4 in the first exon of the TMPRSS2 isoform 1 is particular targeted by the G4 ligand and affects viral replication. Analysis of potential single nucleotide polymorphisms (SNPs) reveals that a reported SNP at this G4 in isoform 1 destroys the G4 motif and makes TMPRSS2 ineffective towards G4 treatment. CONCLUSION: These findings uncover a novel mechanism in which G4 stabilization impacts SARS-CoV-2 replication by changing TMPRSS2 isoform 1 gene expression.

Hemocyte Nuclei Isolation from Adult Drosophila melanogaster for snRNA-seq.

In adult Drosophila, most of the hemocytes are macrophage-like cells (so called plasmatocytes), which serve various functions in organ homeostasis and immune defense. Ontogeny and functions are largely conserved between vertebrate and invertebrate macrophages. Hence, Drosophila offers a powerful genetic toolbox to study macrophage function and genetically modulate these cells. Technological advances in high-throughput sequencing approaches allowed to give an in-depth characterization of vertebrate macrophage populations and their heterogenous composition within different organs as well as changes in disease. Embryonic and larval hemocytes in Drosophila have been recently analyzed in single-cell RNA-sequencing (scRNA-seq) approaches during infection and steady state. These analyses revealed anatomical and functional Drosophila hemocyte subtypes dedicated to specific tasks. Only recently, the Fly Cell Atlas provided a whole transcriptomic single-cell atlas via single-nuclei RNA-sequencing (snRNA-seq) of adult Drosophila including many different tissues and cell types where hemocytes were also included. Yet, a specific protocol to isolate nuclei from adult hemocytes for snRNA-seq and study these cells in different experimental conditions was not available. In this chapter, we give a detailed protocol to purify hemocyte nuclei from adult Drosophila, which can be used in subsequent analyses such as snRNA-seq.

Detection of G-Quadruplex DNA Structures in Macrophages.

In addition to the canonical B-DNA conformation, DNA can fold into different secondary structures. Among them are G-quadruplex structures (G4s). G4 structures are very stable and can fold in specific guanine-rich regions in DNA and RNA. Different in silico, in vitro, and in cellulo experiments have shown that G4 structures form so far in all tested organisms. There are over 700,000 predicted G4s in higher eukaryotes, but it is so far assumed that not all will form at the same time. Their formation is dynamically regulated by proteins and is cell type-specific and even changes during the cell cycle or during different exogenous or endogenous stimuli (e.g., infection or developmental stages) can alter the G4 level. G4s have been shown to accumulate in cancer cells where they contribute to gene expression changes and the mutagenic burden of the tumor. Specific targeting of G4 structures to impact the expression of oncogenes is currently discussed as an anti-cancer treatment. In a tumor microenvironment, not only the tumor cells will be targeted by G4 stabilization but also immune cells such as macrophages. Although G4s were detected in multiple organisms and different cell types, only little is known about their role in immune cells. Here, we provide a detailed protocol to detect G4 formation in the nucleus of macrophages of vertebrates and invertebrates by microscopic imaging.

The telomerase inhibitor imetelstat differentially targets JAK2V617F versus CALR mutant myeloproliferative neoplasm cells and inhibits JAK-STAT signaling.

Imetelstat shows activity in patients with myeloproliferative neoplasms, including primary myelofibrosis (PMF) and essential thrombocythemia. Here, we describe a case of prolonged disease stabilization by imetelstat treatment of a high-risk PMF patient enrolled into the clinical study MYF2001. We confirmed continuous shortening of telomere length (TL) by imetelstat treatment but observed emergence and expansion of a KRAST58I mutated clone during the patient's clinical course. In order to investigate the molecular mechanisms involved in the imetelstat treatment response, we generated induced pluripotent stem cells (iPSC) from this patient. TL of iPSC-derived hematopoietic stem and progenitor cells, which was increased after reprogramming, was reduced upon imetelstat treatment for 14 days. However, while imetelstat reduced clonogenic growth of the patient's primary CD34+ cells, clonogenic growth of iPSC-derived CD34+ cells was not affected, suggesting that TL was not critically short in these cells. Also, the propensity of iPSC differentiation toward megakaryocytes and granulocytes was not altered. Using human TF-1MPL and murine 32DMPL cell lines stably expressing JAK2V617F or CALRdel52, imetelstat-induced reduction of viability was significantly more pronounced in CALRdel52 than in JAK2V617F cells. This was associated with an immediate downregulation of JAK2 phosphorylation and downstream signaling as well as a reduction of hTERT and STAT3 mRNA expression. Hence, our data demonstrate that imetelstat reduces TL and targets JAK/STAT signaling, particularly in CALR-mutated cells. Although the exact patient subpopulation who will benefit most from imetelstat needs to be defined, our data propose that CALR-mutated clones are highly vulnerable.

UV-induced G4 DNA structures recruit ZRF1 which prevents UV-induced senescence.

Senescence has two roles in oncology: it is known as a potent tumor-suppressive mechanism, which also supports tissue regeneration and repair, it is also known to contribute to reduced patient resilience, which might lead to cancer recurrence and resistance after therapy. Senescence can be activated in a DNA damage-dependent and -independent manner. It is not clear which type of genomic lesions induces senescence, but it is known that UV irradiation can activate cellular senescence in photoaged skin. Proteins that support the repair of DNA damage are linked to senescence but how they contribute to senescence after UV irradiation is still unknown. Here, we unraveled a mechanism showing that upon UV irradiation multiple G-quadruplex (G4) DNA structures accumulate in cell nuclei, which leads to the recruitment of ZRF1 to these G4 sites. ZRF1 binding to G4s ensures genome stability. The absence of ZRF1 triggers an accumulation of G4 structures, improper UV lesion repair, and entry into senescence. On the molecular level loss of ZRF1 as well as high G4 levels lead to the upregulation of DDB2, a protein associated with the UV-damage repair pathway, which drives cells into senescence.

A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA.

RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.

RNA-DNA hybrids prevent resection at dysfunctional telomeres.

At critically short telomeres, stabilized TERRA RNA-DNA hybrids drive homology-directed repair (HDR) to delay replicative senescence. However, even at long- and intermediate-length telomeres, not subject to HDR, transient TERRA RNA-DNA hybrids form, suggestive of additional roles. We report that telomeric RNA-DNA hybrids prevent Exo1-mediated resection when telomeres become non-functional. We used the well-characterized cdc13-1 allele, where telomere resection can be induced in a temperature-dependent manner, to demonstrate that ssDNA generation at telomeres is either prevented or augmented when RNA-DNA hybrids are stabilized or destabilized, respectively. The viability of cdc13-1 cells is affected by the presence or absence of hybrids accordingly. Telomeric hybrids do not affect the shortening rate of bulk telomeres. We suggest that TERRA hybrids require dynamic regulation to drive HDR at short telomeres; hybrid presence may initiate HDR through replication stress, whereby their removal allows strand resection.

Prioritization of non-coding elements involved in non-syndromic cleft lip with/without cleft palate through genome-wide analysis of de novo mutations.

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a highly heritable facial disorder. To date, systematic investigations of the contribution of rare variants in non-coding regions to nsCL/P etiology are sparse. Here, we re-analyzed available whole-genome sequence (WGS) data from 211 European case-parent trios with nsCL/P and identified 13,522 de novo mutations (DNMs) in nsCL/P cases, 13,055 of which mapped to non-coding regions. We integrated these data with DNMs from a reference cohort, with results of previous genome-wide association studies (GWASs), and functional and epigenetic datasets of relevance to embryonic facial development. A significant enrichment of nsCL/P DNMs was observed at two GWAS risk loci (4q28.1 (p = 8 × 10-4) and 2p21 (p = 0.02)), suggesting a convergence of both common and rare variants at these loci. We also mapped the DNMs to 810 position weight matrices indicative of transcription factor (TF) binding, and quantified the effect of the allelic changes in silico. This revealed a nominally significant overrepresentation of DNMs (p = 0.037), and a stronger effect on binding strength, for DNMs located in the sequence of the core binding region of the TF Musculin (MSC). Notably, MSC is involved in facial muscle development, together with a set of nsCL/P genes located at GWAS loci. Supported by additional results from single-cell transcriptomic data and molecular binding assays, this suggests that variation in MSC binding sites contributes to nsCL/P etiology. Our study describes a set of approaches that can be applied to increase the added value of WGS data.

Deficiency for SAMHD1 activates MDA5 in a cGAS/STING-dependent manner.

Defects in nucleic acid metabolizing enzymes can lead to spontaneous but selective activation of either cGAS/STING or RIG-like receptor (RLR) signaling, causing type I interferon-driven inflammatory diseases. In these pathophysiological conditions, activation of the DNA sensor cGAS and IFN production are linked to spontaneous DNA damage. Physiological, or tonic, IFN signaling on the other hand is essential to functionally prime nucleic acid sensing pathways. Here, we show that low-level chronic DNA damage in mice lacking the Aicardi-Goutières syndrome gene SAMHD1 reduced tumor-free survival when crossed to a p53-deficient, but not to a DNA mismatch repair-deficient background. Increased DNA damage did not result in higher levels of type I interferon. Instead, we found that the chronic interferon response in SAMHD1-deficient mice was driven by the MDA5/MAVS pathway but required functional priming through the cGAS/STING pathway. Our work positions cGAS/STING upstream of tonic IFN signaling in Samhd1-deficient mice and highlights an important role of the pathway in physiological and pathophysiological innate immune priming.

Detecting G4 unwinding.

DNA can, in addition to the B-DNA conformation, fold into a variety of additional conformations. Among them are G-quadruplex structures that have gained a lot of attention in recent years. G-quadruplex structures (G4s) are highly stable nucleic acid structures that can fold within DNA and RNA molecules. They form in guanine-rich regions that harbor a specific G4 motif. The three-dimensional structure forms via Hoogsteen hydrogen bonding, where the guanines form hydrogen bonds to each other in order to generate G quartets, which stack in order to become G4 structures. The existence and relevance of G4s was controversial as discussed in the past. However, accumulating data was published that supported the model that G4s form in living cells and importantly support biological processes. G4 formation and unfolding is tightly regulated in vivo. If G4s persist in the cell, they can lead to cellular defects such as genome instability. To avoid G4 accumulation in cells, and by this prevent cellular defect, cells has evolved a variety of proteins, mostly helicases, that efficiently unfold G4 DNA and RNA structures. Here, we describe a detailed protocol to monitor G4 structure unfolding by helicases.

Action and function of helicases on RNA G-quadruplexes.

The nucleic acid structure called G-quadruplex (G4) is currently discussed to function in nucleic acid-based mechanisms that influence several cellular processes. They can modulate the cellular machinery either positively or negatively, both at the DNA and RNA level. The majority of what we know about G4 biology comes from DNA G4 (dG4) research. RNA G4s (rG4), on the other hand, are gaining interest as researchers become more aware of their role in several aspects of cellular homeostasis. In either case, the correct regulation of G4 structures within cells is essential and demands specialized proteins able to resolve them. Small changes in the formation and unfolding of G4 structures can have severe consequences for the cells that could even stimulate genome instability, apoptosis or proliferation. Helicases are the most relevant negative G4 regulators, which prevent and unfold G4 formation within cells during different pathways. Yet, and despite their importance only a handful of rG4 unwinding helicases have been identified and characterized thus far. This review addresses the current knowledge on rG4s-processing helicases with a focus on methodological approaches. An example of a non-helicase rG4s-unwinding protein is also briefly described.

Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage.

BACKGROUND: The main function of telomerase is at the telomeres but under adverse conditions telomerase can bind to internal regions causing deleterious effects as observed in cancer cells. RESULTS: By mapping the global occupancy of the catalytic subunit of telomerase (Est2) in the budding yeast Saccharomyces cerevisiae, we reveal that it binds to multiple guanine-rich genomic loci, which we termed "non-telomeric binding sites" (NTBS). We characterize Est2 binding to NTBS. Contrary to telomeres, Est2 binds to NTBS in G1 and G2 phase independently of Est1 and Est3. The absence of Est1 and Est3 renders telomerase inactive at NTBS. However, upon global DNA damage, Est1 and Est3 join Est2 at NTBS and telomere addition can be observed indicating that Est2 occupancy marks NTBS regions as particular risks for genome stability. CONCLUSIONS: Our results provide a novel model of telomerase regulation in the cell cycle using internal regions as "parking spots" of Est2 but marking them as hotspots for telomere addition.

The Relevance of G-Quadruplexes for DNA Repair.

DNA molecules can adopt a variety of alternative structures. Among these structures are G-quadruplex DNA structures (G4s), which support cellular function by affecting transcription, translation, and telomere maintenance. These structures can also induce genome instability by stalling replication, increasing DNA damage, and recombination events. G-quadruplex-driven genome instability is connected to tumorigenesis and other genetic disorders. In recent years, the connection between genome stability, DNA repair and G4 formation was further underlined by the identification of multiple DNA repair proteins and ligands which bind and stabilize said G4 structures to block specific DNA repair pathways. The relevance of G4s for different DNA repair pathways is complex and depends on the repair pathway itself. G4 structures can induce DNA damage and block efficient DNA repair, but they can also support the activity and function of certain repair pathways. In this review, we highlight the roles and consequences of G4 DNA structures for DNA repair initiation, processing, and the efficiency of various DNA repair pathways.

BG-flow, a new flow cytometry tool for G-quadruplex quantification in fixed cells.

BACKGROUND: Nucleic acids can fold into non-canonical secondary structures named G-quadruplexes (G4s), which consist of guanine-rich sequences stacked into guanine tetrads stabilized by Hoogsteen hydrogen bonding, π-π interactions, and monovalent cations. G4 structure formation and properties are well established in vitro, but potential in vivo functions remain controversial. G4s are evolutionarily enriched at distinct, functional genomic loci, and both genetic and molecular findings indicate that G4s are involved in multiple aspects of cellular homeostasis. In order to gain a deeper understanding of the function of G4 structures and the trigger signals for their formation, robust biochemical methods are needed to detect and quantify G4 structures in living cells. Currently available methods mostly rely on fluorescence microscopy or deep sequencing of immunoprecipitated DNA or RNA using G4-specific antibodies. These methods provide a clear picture of the cellular or genomic localization of G4 structures but are very time-consuming. Here, we assembled a novel protocol that uses the G4-specific antibody BG4 to quantify G4 structures by flow cytometry (BG-flow). RESULTS: We describe and validate a flow cytometry-based protocol for quantifying G4 levels by using the G4-specific antibody BG4 to label standard cultured cells (Hela and THP-1) as well as primary cells obtained from human blood (peripheral blood mononuclear cells (PBMCs)). We additionally determined changes in G4 levels during the cell cycle in immortalized MCF-7 cells, and validated changes previously observed in G4 levels by treating mouse macrophages with the G4-stabilizing agent pyridostatin (PDS). CONCLUSION: We provide mechanistic proof that BG-flow is working in different kinds of cells ranging from mouse to humans. We propose that BG-flow can be combined with additional antibodies for cell surface markers to determine G4 structures in subpopulations of cells, which will be beneficial to address the relevance and consequences of G4 structures in mixed cell populations. This will support ongoing research that discusses G4 structures as a novel diagnostic tool.

G-quadruplexes: a promising target for cancer therapy.

DNA and RNA can fold into a variety of alternative conformations. In recent years, a particular nucleic acid structure was discussed to play a role in malignant transformation and cancer development. This structure is called a G-quadruplex (G4). G4 structure formation can drive genome instability by creating mutations, deletions and stimulating recombination events. The importance of G4 structures in the characterization of malignant cells was currently demonstrated in breast cancer samples. In this analysis a correlation between G4 structure formation and an increased intratumor heterogeneity was identified. This suggests that G4 structures might allow breast cancer stratification and supports the identification of new personalized treatment options. Because of the stability of G4 structures and their presence within most human oncogenic promoters and at telomeres, G4 structures are currently tested as a therapeutic target to downregulate transcription or to block telomere elongation in cancer cells. To date, different chemical molecules (G4 ligands) have been developed that aim to target G4 structures. In this review we discuss and compare G4 function and relevance for therapeutic approaches and their impact on cancer development for three cancer entities, which differ significantly in their amount and type of mutations: pancreatic cancer, leukemia and malignant melanoma. G4 structures might present a promising new strategy to individually target tumor cells and could support personalized treatment approaches in the future.